CN111304292A - Gene fine screening method beneficial to pig breeding - Google Patents
Gene fine screening method beneficial to pig breeding Download PDFInfo
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- CN111304292A CN111304292A CN202010315468.5A CN202010315468A CN111304292A CN 111304292 A CN111304292 A CN 111304292A CN 202010315468 A CN202010315468 A CN 202010315468A CN 111304292 A CN111304292 A CN 111304292A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 64
- 238000009395 breeding Methods 0.000 title claims abstract description 33
- 230000001488 breeding effect Effects 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000012216 screening Methods 0.000 title claims abstract description 20
- 230000009286 beneficial effect Effects 0.000 title claims abstract description 18
- 238000012408 PCR amplification Methods 0.000 claims abstract description 18
- 208000035240 Disease Resistance Diseases 0.000 claims abstract description 16
- 239000000463 material Substances 0.000 claims abstract description 11
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 claims abstract description 9
- 230000002349 favourable effect Effects 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 4
- 241000282887 Suidae Species 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 238000004458 analytical method Methods 0.000 claims description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 238000012300 Sequence Analysis Methods 0.000 claims description 3
- 108010006785 Taq Polymerase Proteins 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 230000003902 lesion Effects 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 238000005215 recombination Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 108700005078 Synthetic Genes Proteins 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000001850 reproductive effect Effects 0.000 description 5
- 239000012634 fragment Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 208000002254 stillbirth Diseases 0.000 description 1
- 231100000537 stillbirth Toxicity 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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Abstract
The invention provides a gene excellent screening method beneficial to pig breeding, and relates to the technical field of biomolecules. The excellent gene screening method favorable for pig breeding comprises the steps of selecting test materials, collecting DNA samples, obtaining primer pairs, carrying out PCR amplification, carrying out polymorphism detection and artificially synthesizing genes. By extracting a DNA sample of a sow and carrying out PCR amplification and RFLP analysis, two excellent genes with high litter size and strong disease resistance which are beneficial to pig breeding can be directly obtained, the speed of obtaining the excellent genes is higher, the probability of obtaining the excellent genes is higher, the difficulty of screening the genes which are beneficial to pig breeding is greatly reduced, and the two excellent genes with high litter size and strong disease resistance can be synthesized together by a synthetic gene technology, so that the two genes can be inherited in a pig breeding system at the same time, and the screened genes are more beneficial to pig breeding.
Description
Technical Field
The invention relates to the technical field of biological molecules, in particular to a gene excellent screening method beneficial to pig breeding.
Background
The pig industry is an important industry in agriculture in China, and plays an important role in guaranteeing safe supply of meat food, the traditional pig industry is changed into the modern pig industry in China at present, the breeding mode, the regional layout, the production mode and the production capacity are obviously changed, and various challenges of weak independent innovation capacity, prominent food safety problem, high labor cost, import dependence of breeders, serious epidemic diseases, high environmental protection pressure, lack of feed resources and the like still exist, but opportunities of improvement of independent innovation conditions, large international market space, steady increase of domestic market, high government support force and the like exist.
In the pig raising industry, the reproductive traits of the sows, such as total litter size, piglet uniformity, stillbirth number, mummy number and the like, have important significance on the economic benefit of sow production, the reproductive traits of the sows are continuously improved to improve the reproductive performance of the sows, the reproductive traits are particularly important in actual production, and statistics of students in the beginning of the 21 st century can show that the economic benefit is averagely increased by about 100 yuan along with the increase of the litter size of the sows by 1.
The reproductive traits are important economic traits for pig production, the heritability is low, the time for obtaining excellent genes beneficial to pig reproduction by a conventional breeding method is long, and great genetic progress is difficult to obtain.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for screening excellent genes beneficial to pig breeding, and solves the problems that the screening of excellent genes beneficial to pig breeding by a conventional breeding method needs a long time and excellent genes are difficult to obtain.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a gene excellent screening method beneficial to pig breeding comprises the following steps:
s1 selection of test materials
Firstly, 800 Sujiang sow sows are selected as test materials;
s2 collecting DNA sample
Extracting DNA samples of ear tissues from each Sujiang sow, dividing the DNA samples into two equal parts, and storing the two parts at the temperature of between 20 ℃ below zero and 80 ℃ below zero;
s3 obtaining primer pair
Performing clustering analysis according to the genes with known large litter size of Meishan pigs to obtain a first primer pair, and performing clustering analysis according to the genes with known strong disease resistance of Changbai pigs to obtain a second primer pair;
s4 PCR amplification
Taking the two extracted DNA samples as templates, and respectively carrying out PCR amplification under the guidance of a first primer pair and a second primer pair to obtain two PCR amplification products;
s5 polymorphism detection
Carrying out polymorphic sequence analysis on the two PCR amplification products by using an RFLP technology, and counting the analysis result to obtain a gene with a large litter size of Sujiang pigs and a gene with strong disease resistance;
s6 artificially synthesized gene
By utilizing the principle of gene recombination, the gene with large litter size of Sujiang pigs and the gene with strong disease resistance are artificially synthesized.
Preferably, the PCR reaction system is: 11.5. mu.l of Taq DNA polymerase, 1.5. mu.l of 50 ng/. mu.l of DNA sample template, 1.0. mu.l of 10pmol/L of the first primer pair or the second primer pair, and then supplementing the reaction system to 25. mu.l with ultrapure water.
Preferably, the PCR reaction conditions are: pre-denaturation at 95 ℃ for 4.5min, denaturation at 95 ℃ for 30s, annealing at 55.5 ℃ for 30s, extension at 71 ℃ for 30s, 35 cycles, and extension at 71 ℃ for 4.5 min.
Preferably, the RFLP reaction system is as follows: mu.l of PCR amplification product is added with 1.0 mu.l of restriction enzyme and 2.0 mu.l of 10 Xbuffer diluent, the reaction system is supplemented with 4.0 mu.l of ultrapure water to 15 mu.l, then the reaction system is heated in water bath for 3.5h at 35-37 ℃ by a water bath kettle, and finally the enzyme digestion result is detected by 2.0% agarose gel electrophoresis.
Preferably, the 800 Sujiang sow pigs are all healthy and disease-free experimental materials.
Preferably, the breeding shape of the sow mainly comprises the litter size, the effective number of the live piglets, the piglet birth lesion coefficient and the like.
(III) advantageous effects
The invention provides a gene excellent screening method beneficial to pig breeding. The method has the following beneficial effects:
1. according to the invention, by extracting the DNA sample of the sow and carrying out PCR amplification and RFLP analysis, two excellent genes with high litter size and strong disease resistance which are beneficial to pig breeding can be directly obtained, compared with the conventional breeding method, the speed of obtaining the excellent genes is higher, the probability of obtaining the excellent genes is higher, and the difficulty of screening the genes which are beneficial to pig breeding is greatly reduced.
2. The invention can synthesize two excellent genes of high litter size and strong disease resistance together by a gene artificial synthesis technology, so that the two genes of high litter size and strong disease resistance can be inherited in a pig breeding system at the same time, and the offspring bred by pigs can have the characteristics of high litter size and strong disease resistance, and the screened gene is more beneficial to the breeding of the pigs.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b):
the embodiment of the invention provides a gene excellent screening method beneficial to pig breeding, which comprises the following steps:
s1 selection of test materials
Firstly, 800 Sujiang sow sows are selected as test materials, and the number of 800 Sujiang sow sows is selected to increase the number of extracted DNA samples, so that the accuracy of test data is ensured;
s2 collecting DNA sample
Extracting DNA samples of ear tissues from each Sujiang sow, dividing the DNA samples into two equal parts, extracting 400 DNA samples in each part by using a DNA extraction kit, and storing at-20 ℃, so that the DNA samples can be better stored, and the samples are prevented from being damaged to cause gene screening failure;
s3 obtaining primer pair
Performing clustering analysis according to genes with known large litter size of Meishan pigs to obtain a first primer pair, wherein the Meishan pigs have the characteristic of large litter size, and performing clustering analysis according to genes with known strong disease resistance of Changbai pigs to obtain a second primer pair, wherein the Changbai pigs have the characteristic of strong disease resistance;
s4 PCR amplification
Taking the two extracted DNA samples as templates, respectively carrying out PCR amplification under the guidance of a first primer pair and a second primer to obtain two PCR amplification products, and increasing the number of genes to be amplified to several million times within 2 to 3 hours through PCR;
s5 polymorphism detection
Carrying out polymorphic sequence analysis on the two PCR amplification products by using an RFLP technology, and counting the analysis result to obtain a gene with a large litter size of Sujiang pigs and a gene with strong disease resistance, wherein the RFLP technology is implemented by the steps of firstly carrying out enzyme digestion on a DNA sample by using restriction enzyme, then separating DNA fragments by using gel electrophoresis, then transferring the DNA fragments to a filter membrane, and finally carrying out hybridization by using a radioactive labeled probe to display the specific DNA fragments and result analysis;
s6 artificially synthesized gene
By utilizing the principle of gene recombination, the gene with large litter size of Sujiang pigs and the gene with strong disease resistance are artificially synthesized, and the two genes are artificially synthesized together, so that the two genes can be inherited in a pig breeding system at the same time, and the stability of excellent gene inheritance favorable for pig breeding is enhanced.
The PCR reaction system is as follows: 11.5. mu.l of Taq DNA polymerase, 1.5. mu.l of 50 ng/. mu.l of DNA sample template, 1.0. mu.l of 10pmol/L of the first primer pair or the second primer pair, and then supplementing the reaction system to 25. mu.l with ultrapure water.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 4.5min, denaturation at 95 ℃ for 30s, annealing at 55.5 ℃ for 30s, extension at 71 ℃ for 30s, 35 cycles, and extension at 71 ℃ for 4.5 min.
The RFLP reaction system is as follows: mu.l of PCR amplification product was added with 1.0. mu.l of restriction enzyme and 2.0. mu.l of 10 XBuffer diluent, and then the reaction system was supplemented with 4.0. mu.l of ultrapure water to 15. mu.l, and then heated in water bath at 37 ℃ for 3.5 hours in a water bath, and finally the cleavage result was detected by 2.0% agarose gel electrophoresis.
The 800 Sujiang sows are all healthy and disease-free experimental materials, so that the diseased pigs are prevented from influencing the experimental materials, and the gene screening fails.
The breeding shape of the sow mainly comprises the litter size, the effective number of the live piglets, the birth lesion coefficient of the piglets and the like.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A gene excellent screening method beneficial to pig breeding comprises the following steps:
s1 selection of test materials
Firstly, 800 Sujiang sow sows are selected as test materials;
s2 collecting DNA sample
Extracting DNA samples of ear tissues from each Sujiang sow, dividing the DNA samples into two equal parts, and storing the two parts at the temperature of between 20 ℃ below zero and 80 ℃ below zero;
s3 obtaining primer pair
Performing clustering analysis according to the genes with known large litter size of Meishan pigs to obtain a first primer pair, and performing clustering analysis according to the genes with known strong disease resistance of Changbai pigs to obtain a second primer pair;
s4 PCR amplification
Taking the two extracted DNA samples as templates, and respectively carrying out PCR amplification under the guidance of a first primer pair and a second primer pair to obtain two PCR amplification products;
s5 polymorphism detection
Carrying out polymorphic sequence analysis on the two PCR amplification products by using an RFLP technology, and counting the analysis result to obtain a gene with a large litter size of Sujiang pigs and a gene with strong disease resistance;
s6 artificially synthesized gene
By utilizing the principle of gene recombination, the gene with large litter size of Sujiang pigs and the gene with strong disease resistance are artificially synthesized.
2. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the PCR reaction system is as follows: 11.5. mu.l of Taq DNA polymerase, 1.5. mu.l of 50 ng/. mu.l of DNA sample template, 1.0. mu.l of 10pmol/L of the first primer pair or the second primer pair, and then supplementing the reaction system to 25. mu.l with ultrapure water.
3. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 4.5min, denaturation at 95 ℃ for 30s, annealing at 55.5 ℃ for 30s, extension at 71 ℃ for 30s, 35 cycles, and extension at 71 ℃ for 4.5 min.
4. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the RFLP reaction system is as follows: mu.l of PCR amplification product is added with 1.0 mu.l of restriction enzyme and 2.0 mu.l of 10 Xbuffer diluent, the reaction system is supplemented with 4.0 mu.l of ultrapure water to 15 mu.l, then the reaction system is heated in water bath for 3.5h at 35-37 ℃ by a water bath kettle, and finally the enzyme digestion result is detected by 2.0% agarose gel electrophoresis.
5. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the 800 Sujiang sow sows are all healthy disease-free experimental materials.
6. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the breeding shape of the sow mainly comprises the litter size, the effective number born, the piglet birth lesion coefficient and the like.
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| CN202010315468.5A CN111304292A (en) | 2020-04-21 | 2020-04-21 | Gene fine screening method beneficial to pig breeding |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992018651A1 (en) * | 1991-04-19 | 1992-10-29 | Iowa State University Research Foundation, Inc. | Genetic markers for pig litter size |
| CN101570789A (en) * | 2009-06-12 | 2009-11-04 | 华中农业大学 | Identification and application of pig MHC II TA gene as immunity related molecular labels |
| CN101828539A (en) * | 2010-05-19 | 2010-09-15 | 扬州大学 | Method for breeding Sujiang pig variety |
| CN107574250A (en) * | 2017-07-23 | 2018-01-12 | 华中农业大学 | A kind of examination and application of the molecular labeling related to characters of number born of sow |
-
2020
- 2020-04-21 CN CN202010315468.5A patent/CN111304292A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992018651A1 (en) * | 1991-04-19 | 1992-10-29 | Iowa State University Research Foundation, Inc. | Genetic markers for pig litter size |
| CN101570789A (en) * | 2009-06-12 | 2009-11-04 | 华中农业大学 | Identification and application of pig MHC II TA gene as immunity related molecular labels |
| CN101828539A (en) * | 2010-05-19 | 2010-09-15 | 扬州大学 | Method for breeding Sujiang pig variety |
| CN107574250A (en) * | 2017-07-23 | 2018-01-12 | 华中农业大学 | A kind of examination and application of the molecular labeling related to characters of number born of sow |
Non-Patent Citations (1)
| Title |
|---|
| 王宵燕等: "ESR 基因在苏姜猪世代选育中的遗传变异及与猪群繁殖性状的关联", 《中国农业科学》 * |
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Application publication date: 20200619 |